This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Characterisation of the equine macrophage / monocyte Anna Eleonora Karagianni Doctor of Philosophy – The University of Edinburgh 2014 Contents Declaration ................................................................................................................... i Dedication ................................................................................................................... ii Acknowledgements .................................................................................................... iii List of relevant publications and presentations ..................................................... vi Abstract ...................................................................................................................... ix Abbreviations ............................................................................................................ xi Chapter 1: Introduction ............................................................................................ 1 1.1 Macrophage biology and the mononuclear phagocyte system .................... 1 1.1.1 Important factors of haemopoietic regulation .......................................... 5 1.2 Markers of the mononuclear phagocyte system ........................................... 7 1.3 Monocyte subsets ....................................................................................... 11 1.4 Macrophage activation ............................................................................... 14 1.4.1 The phenomenon of endotoxin tolerance ............................................... 18 1.5 Lung macrophages and their microenvironment ........................................ 20 1.5.1 Lung microenvironment ......................................................................... 20 1.5.2 Phenotype and function of alveolar macrophages in comparison with macrophages/monocytes from different anatomical locations in humans and mice……. ........................................................................................................... 22 1.6 The equine macrophage and the immune system ...................................... 25 1.6.1 Equine bone marrow derived and monocyte derived macrophages ...... 25 1.6.2 Equine peritoneal macrophages ............................................................. 27 1.6.3 Macrophages of the equine lung ............................................................ 27 1.7 Horse as an animal model for human disease research .............................. 30 1.7.1 Genome and microarray analysis in the horse ....................................... 34 1.8 The horse as an elite athlete ....................................................................... 36 1.8.1 Pathophysiological changes during exercise .......................................... 37 1.8.2 Exercise immunology ............................................................................ 38 1.9 Inflammatory Airway Disease ................................................................... 50 1.10 Preventive measures for exercise induced dysregulation of the immune system 55 1.10.1 Dietary measures ................................................................................ 55 1.10.2 Macrophage regulation....................................................................... 55 Doctor of Philosophy – The University of Edinburgh 2014 1.11 Rationale and aims of the study ................................................................. 58 Chapter 2: Materials and methods ......................................................................... 60 2.1 Animals used in the study .......................................................................... 60 2.1.1 Euthanised horses ................................................................................... 60 2.1.2 Standardbred racehorses ........................................................................ 61 2.2 Sample collection and cell isolation ........................................................... 63 2.2.1 Bronchoalveolar lavage fluid (BALF) ................................................... 63 2.2.2 Peritoneal lavage fluid (PLF) ................................................................. 64 2.2.3 Cell isolation from BALF and PLF ........................................................ 64 2.2.4 Spleen cell isolation ............................................................................... 65 2.2.5 Isolation of peripheral blood mononuclear cells (PBMCs) ................... 65 2.2.6 Cryopreservation .................................................................................... 66 2.3 Cell culture ................................................................................................. 66 2.4 Enzyme linked immunosorbent assay ........................................................ 67 2.5 Nitrite assay ................................................................................................ 68 2.6 Flow cytometry assay ................................................................................. 69 2.6.1 CD14, TLR4 and CD163 cell surface expression .................................. 69 2.6.2 Phagocytosis assay ................................................................................. 70 2.7 RNA analysis ............................................................................................. 71 2.7.1 Total RNA extraction ............................................................................. 71 2.7.2 RNA quality assessment ........................................................................ 72 2.7.3 cDNA synthesis ...................................................................................... 72 2.8 Real time quantitative polymerase chain reaction (RT qPCR) .................. 73 2.8.1 Primer efficiency .................................................................................... 75 2.8.2 Reference gene selection ........................................................................ 76 2.9 Statistical analysis ...................................................................................... 77 2.10 Microarray analysis .................................................................................... 77 2.10.1 Partek microarray analysis ................................................................. 78 2.10.2 Biolayout microarray analysis............................................................ 79 2.10.3 Functional annotation ......................................................................... 80 Chapter 3: Extensive studies on the equine macrophage/monocyte ................... 81 3.1 Establishing a protocol for horse macrophage isolation and activation ..... 81 Doctor of Philosophy – The University of Edinburgh 2014 3.1.1 Monocytes / macrophages can be isolated in high yield from the lungs, peritoneal cavity, spleen and peripheral blood from a single horse. .................. 81 3.1.2 Cryopreserved monocytes/macrophages can be recovered from storage and retain their activity ...................................................................................... 84 3.1.3 Differential cell counts on alveolar and peritoneal lavage ..................... 85 3.1.4 Alveolar macrophages differ from peritoneal macrophages with respect to their morphology ............................................................................................ 90 3.1.5 The response of equine macrophages to LPS is dependent on the type of serum used in the culture medium ..................................................................... 93 3.1.6 Cryopreserved AMs and PMs, following LPS stimulation, show a similar pattern of cytokine response as the freshly harvested cells ................... 98 3.1.7 In contrast to LPS treated mouse macrophages, equine AMs and PMs did not produce NO .......................................................................................... 102 3.1.8 Equine AMs and blood monocytes show endotoxin tolerance ............ 106 3.1.9 rhCSF1 and high concentration of HS induced differentiation of horse PBMCs to macrophages ................................................................................... 110 3.1.10 CD14, CD163 and TLR4 are present on equine blood monocytes ...... 113 3.2 Equine alveolar macrophages differ in their function and phenotype from peritoneal macrophages ........................................................................................ 116 3.2.1 AMs, but not PMs, respond to different inflammatory stimuli ............ 116 3.2.2 Identification of suitable reference genes for RT qPCR assay in AMs 120 3.2.3 LPS-induced TNFα and IDO mRNA expression in AMs and PMs ..... 122 3.2.4 CD14, CD163 and TLR4 expression in AMs